[Gann, 74, 437-444; June, 1983]

ANTIUMOR EFFECT AND METABOLIC ACTIVATION OF CYCLOPHOSPHAMIDE

AND 4-HYDROPEROXYCYCLOPHOSPHAMIDE IN THE HUMAN BREAST CARCINOMA (MX-1)-NUDE MOUSE SYSTEM

Tetsuro KUBOTA,*1,*3Yuji HANATANI,*1 Ken TsuYUKI,*1 Munehiko NAKADA,*1 Kyuya IsxnBIKI,*1 Osahiko ABE,*1 Tetsuya KAMATAKI*2 and Ryuichi KATO*2 *1Department of Surgery and *2Department of Pharmacology, School of Medicine, Keio University, Shinanomachi 35, Shinjuku-ku, Tokyo 160

The effects of cyclophosphamide (CPA) and its active form, 4-hydroperoxy-CPA,

against human breast carcinoma transplanted into nude mice (BALB/c •ôNH•ônu/nu•ôNS•ô)were evaluated in terms of the decreases of hepatic drug-metabolizing enzymes in nude

mice. A human breast carcinoma, MX-1, was implanted into the subcutaneous tissue of nude mice and a drug was administered intravenously once at a dose of 0.05, 0.1 or 0.15 mmol/kg, 1 or 3 weeks after tumor inoculation. 4-Hydroperoxy- CPA was more effective than CPA as regards inhibition of tumor growth, and the

difference in effect was greater when the drugs were administered 3 weeks after tumor inoculation. The activity of CPA was depressed by the decrease of the hepatic

drug-metabolizing enzymes in proportion to the tumor-bearing period. Therefore, the effects of masked derivatives of CPA may correlate with the changes in drug metabolizingactivities of tumor-bearing mice. The human tumor xenografts-nude mice system is considered to be suitable for chemosensitivity tests with masked compounds.

Key words: Human breast carcinoma (MX-1)-Nude mice - Masked com- pounds-Cytochrome P-450-Drug activation

Since experimental cancer chemotherapy mycin,cyclophosphamide, 3-[(4-amino-2- against human tumor xenografts serially methyl-5-pyrimidinyl) methyl]-1-(2-chloro transplanted into nude mice was reported ethyl)-1-nitrosourea (ACNU), and tegafur by Povlsen and Rygaard,28) many papers against human tumors serially transplanted have been published to demonstrate that into nude mice.5,17-20)Although masked com this human tumor xenografts-nude mice poundsare considered to be metabolized in system is valuable for the evaluation of the nude mice on the grounds that cyclophos effects of anticancer agents on human phamideand tegafur were found to be effec tumors.25,30,32) It was expected that masked tiveon human tumors,17-19)the effects of the compounds which were ineffective in vitro metabolizing activities of nude mice on the could also be evaluated in this experimental antitumor action of masked compounds have system. Since 1976 , we have reported the not been sufficiently elucidated. antitumor activities of several compounds, It is well known that the activities of including mitomycin C , aclarubicin, adria drug-metabolizing enzymes of liver micro *3 To whom requests for reprints should be somesare markedly decreased in tumor addressed. Present address: Department of Sur bearingrats,11-14) and that drug metabolism gery,the Kitasato Institute Hospital, Shirokane is impaired in mice bearing various trans 5-9-1, Minato-ku , Tokyo 108. plantabletumors.1,23,29) Recently, we have

74(3) 1983 437 T. KUBOTA, ET AL.

observed a decrease in the activities of of cyclophosphamide oxidase was determined ac hepatic drug-metabolizing enzymes in nude cordingto the methods of Sladek.33) mice bearing human tumor xenografts and Drug Cyclophosphamide and its active form, 4-hydroperoxy-CPA, were used for the experi

syngeneic murine tumors.15) ments.These drugs were provided by Shionogi The purpose of the present paper was to and Co., Ltd., Osaka. A single dose of 0.05, 0.1 or compare the relative potency of antitumor 0.15 mmol of the drug per kg body weight dissolved activities of cyclophosphamide (CPA) and in 0.2 ml of physiological saline was administered intravenously at 1 or 3 weeks after tumor inocu 4-hydroperoxy-CPA, which does not require lation.At the time of treatment, tumor-bearing any metabolic activation, against trans mice were randomized into test groups consisting plantedhuman tumors in nude mice in of four mice each. relation to the drug-metabolizing activities. Tumor Inoculation, Measurement of Tumor Size, and Evaluation of Drug Activity One MATERIALSAND METHODS tumor tissue fragment of approximately 3•~3•~3 mm in size was implanted into the subcutaneous Mice Nude mice with a BALB/cgenetic back tissue of the backs of mice under ether anesthesia groundwere bred under specific pathogen-free by means of a trocar needle. Tumors were mea conditions at the Central Institute for Experi sured(lengh and width) with a sliding caliper mentalAnimals (Kawasaki),and were then main three times weekly by the same person. tainedin autoclaved cap cages with autoclaved According to the Battelle Memorial Institute food, water, and bedding in the Experimental Protocol (the National Cancer Institute, U.S.A.),5, Animal Center of our Institute. Six-to seven-week 27) the tumor weight (W) in mg was calculated oldmale nude mice weighing 20-22g were used from the linear measurements by using the for for the experiments. mulaTumor weight (mg)=length (mm)•~[width Tumor A human breast carcinoma,MX-1, se (mm)]2/2, as described in Cancer Chemother. Rep., riallytransplanted into nude mice was used. MX-1 Part 3, 3, 1-103 (1972). The relative mean tumor was establishedin 1974 by Giovanella27)from the weight was calculated as RW=W1/W0, where cancerous tissue of a premenopausal 29-year-old W1 is the mean tumor weight of a group at any female who had not been given any antitumor given time and W0 is the mean tumor weight at agents. This tumor was kindly supplied by Dr. K. the time of treatment. The results were evaluated Inoue,8) Cancer Chemotherapy Center, Tokyo. by the method of Geran et al.4) as follows: ++, The histological features of MX-1 are those of regression of tumor RW<1.0; +, retardation of undifferentiatedcarcinoma consistingof polygonal tumor growth TRW/CRW?42%; -, inactive cellswith rather uniform round nuclei and scanty TRW/CRW>42%, where TRW is the relative cytoplasmaand scanty interstitial tissue. mean tumor weight of the treated group and CRW The tumor take rate was 98.9% (89/90) and the is the relative mean tumor weight of the control availability rate for experiments was 93.9% group. (84/90). The mass doubling time during the ex The coefficients of correlation between anti ponentialgrowth phase was 4.7 days and the tumoreffects in terms of the lowest value of TRW/ labelingindex at the same period was 23.9%. CRW and doses of drug in terms of mmol/kg were Assay of Liver Microsomal Drug-metab calculated. Student's t-test was applied to confirm olizingEnzymes Enzyme assays were per the statistical significance of the results. The re formedin MX-1-bearingnude mice at 1, 2 and 3 gressionequation was calculated as y=a-bx, weeks after tumor inoculations.The experimental where y is the value of log TRW/CRW and x is the and control groups all consistedof 6 mice. dose in mmol/kg. As the value of a was close to 2.0 After sacrifice of the animals, the livers were in all four equations, the value of b was used to immediately removed and homogenized with 3 compare the effect of drugs, i.e., the larger the volumes of ice-cold 1.15% KCl solution in a Te value of b, the greater the drug effectiveness. flon-glasshomogenizer. After centrifugationof the

homogenatesat 9,000gfor 20min, the supernatant RESULTS fractions were centrifugedat 105,000gfor 1hr and the soluble fractions were decanted. The micro somalpellets were rinsed and resuspendedin ice Changes in Activity of Drug-metaboliz cold1.15% KCl solution. The amounts of micro ingEnzymes Table I shows body weights, somalprotein and P-450 were determined as de liver weights, spleen weights, tumor weights,

scribedin a previous paper,9,26)and the activity contents of microsomal proteins and cyto

- 438 Gann EFFECT OF P-450 ON DRUG ACTIVATION chrome P-450, and CPA activation in tumor Drug Activity The effects of CPA and bearingand tumor-free mice. No statistically 4-hydroperoxy-CPA on MX-1 are sum significant changes were observed in body marizedin Table II. Tumor regressions and liver weights. Although some weight were observed in all experimental groups, loss was noted when the body weight was except in the group receiving 0.05 mmol corrected by deducting the tumor weight, no of CPA per kg when drugs were adminnis wasting syndrome developed during the tered1 week after tumor inoculation. How experiments. Spleen weight was found to ever,all the tumors regrew within two weeks be increased in nude mice having trans after drug administration. On the other plantedMX-1, with statistical significance. hand, when the treatment was performed Tumor weight increased exponentially from 3 weeks after tumor inoculation, no tumor 1 to 3 weeks after tumor inoculation. The regressions were observed except in the content of liver microsomal protein was groups given 0.1 and 0.15 mmol of 4-hy slightly decreased depending on the tumor droperoxy-CPA per kg. Even though 0.05 bearingperiod, and a statistically significant mmol of CPA per kg was ineffective, the decrease was found at 3 weeks. The content same dose of 4-hydroperoxy-CPA produced of cytochrome P-450 was not decreased at tumor regression when it was administered 1 or 2 weeks but was depressed at 3 weeks 1 week after tumor inoculation. Both drugs after tumor inoculation. Although CPA were more effective when the treatment was activation was not depressed 1 week after given 1 week after tumor inoculation. inoculation, a statistically significant de No statistically significant decrease of creasewas found at 2 and 3 weeks after body weight was observed during any of tumor inoculation. Thus, microsomal drug the experiments, indicating that these doses metabolizingenzymes were found to be of CPA and 4-hydroperoxy-CPA were not depressed dependently upon the tumor toxic to mice. No decrease of spleen weight bearingperiod. The content of cytochrome was noticed except in one group given CPA, P-450 was depressed to almost half and suggesting that bone marrow suppression CPA activation to almost 40% of those of by CPA and 4-hydroperoxy-CPA was not control nude mice. significant at these doses.

Table I. Activities of the Hepatic Microsomal Drug-metabolizing Enzyme System in Tumor-bearing Nude Mice

Mice were implanted subcutaneously with MX-1 (undifferentiated human carcinoma of the breast) and sacrificed after 1, 2, and 3 weeks. *P<0 .05, **P<0.01, ***P<0.001. 74(3) 1983 439 T. KUBOTA, ET AL.

Table II. Effects of CPA and 4-Hydroperoxy-CPA on MX-1

a) Weeks after tumor inoculation. b) The lowest value during the experiments. * P<0 .05

Table III. Correlation between Antitumor Effect and Drug Dose

a) Weeks after tumor inoculation. b) Coefficient of correlation between log TRW/CRW*and mmol/kg. c) Y=log TRW/CRW*, x=mmol/kg. * The lowest value during the experiments .

The dose-response relation for antitumor - CPA was more effective than CPA at the effect is shown in Table III. In all four same administration time, and the difference groups, an exponential relationship was between CPA and 4-hydroperoxy-CPA was observed between the dosage of drugs in greater at 3 weeks (bOOH-bCPA=4.67) mmol/kg and the antitumor effect in terms than at 1 week (bOOH-bCPA=2.23). (The of TRW/CRW.The coefficients of correlation subscript OOH refers to 4-hydroperoxy- ranged from-0.969 to-0.997 and were CPA.) statistically significant (P<0.05). From the regression equations of the correlations, DISCUSSION it was found that administration at 1 week Masked compounds, including cyclophos was more effective than administration at phamide,tegafur,16) and carmofur,6) have 3 weeks with both CPA (b1-b3=7.46) and often been utilized in cancer treatments, 4-hydroperoxy-CPA (b1-b3=5.02), and the especially as a chemotherapy adjuvant to difference between administration at 1 and surgical operations. These compounds make 3 weeks was larger in the case of CPA (7.46> long-term post-operative adjuvant chemo 5.02). It was also found that 4-hydroperoxy therapypossible for outpatients.10) As these

440 Gann EFFECT OF P-450 ON DRUG ACTIVATION compounds need to be activated by micro livers and kidneys of homozygous nude somaldrug-metabolizing enzymes to exhibit Swiss, heterozygous normal Swiss, homo antitumor effects, it is difficult to evaluate zygousnormal Swiss and DBA/2 mice and their effectivenessin cultured human tumor reported that the hepatic cytochrome P- cells in vitro. 450 concentrations were all similar, except Since successful transplantation of human for DBA/2 mice, which had a significantly tumor cells into nude mice was reported,28) lower concentration. Litterst et al.24)also ob this human tumor xenografts-nude mice servedno substantial difference in amounts system has been used for experimental can of cytochrome P-450 among nude mice, cerchemotherapy in vivo.25,30,32)Although normal heterozygous littermates, and the cyclophoshamide and tegafur have been wild strain. Although our values of cyto reported to exhibit some effects on human chromeP-450 are slightly lower than those tumors transplanted into nude mice,18,19) of Freudenthal et al.,2) no significant dif their modalities of activation were not ferenceswere observed between nude mice fully clarified. As new masked compounds with the NIH Swiss and BALB/c genetic will be developed in future, a screening backgrounds. From these results, the nu system to evaluate their efficacy is needed. gene was thought to have little effect on the Thus, it was thought to be important to drug-metabolizing enzymes. It was also sug elucidate the drug-metabolizing activities gestedthat the decrease in the activity of of nude mice and their influence on the drug-metabolizing enzymes was not caused action of masked compounds against human by the immunological reactions of tumor tumor xenografts. bearinganimals.15) MX-1 originated from a premenopausal Microsomal proteins, contents of cyto 29-year-old female without any prior cancer chromeP-450, and CPA activation were chemotherapy. The patient did not respond depressed, depending on the duration of to radiation or 5-fluorouracil (5-FU) and the tumor-bearing period. Since the tumor died six months later.27) The experimental size increased in relation to the tumor-bear data in nude mice also indicated that MX-1 ingperiod, the possibility arose that the was insensitive to 5-FU.7,27) This correla decrease of enzymes depended on the tumor tionbetween clinical data and experimental size. However, in the provious paper,15) results corresponded well to previous re we showed that the decrease of drug-metab- ports.3,18)Although CPA had not been olizing activity was probably dependent on administered to the donor patient, this the duration of the tumor-bearing period agent was evaluated as effective against rather than the size of the tumor. MX-1 by Ovejera et al.27)and Inoue et al.8) CPA is thought to exhibit antitumor This susceptibility was confirmed by our action after being metabolized to 4-hydroxy- experiments, which revealed that the chemo CPA by P-450 in the presence of NADPH sensitivityof this strain had been retained and oxygen.22) However, as 4-hydroxy-CPA for five years. Further, it was reported that is unstable, 4-hydroperoxy-CPA was used the effect of alkylating agents on MX-1 as an active form of CPA. 4-Hydroperoxy- corresponded well to clinically observed pat CPA is easily converted to 4-hydroxy-CPA ternsof chemosensitivity in patients with in vivo, and thus it produces antitumor breast cancer.8) On the basis of these con effect without being metabolized by cyto siderations,this tumor was thought to be chromeP-450.34) 4-Hydroperoxy-CPA is suitable as a human tumor model. more effective than CPA at a given dose Freudenthal et al.2) compared the activ (in mmol/kg). This might be due to the ityof drug-metabolizing enzymes in the fact that not all CPA can be metabolized 74(3) 1983 441 T. KUBOTA, ET AL.

to the active form and some CPA is excreted before metabolic activation.22) In the present ACKNOWLEDGMENTS

studies, we observed that CPA was markedly This work was supported in part by a Grant-in effective at 1 week but less effective at 3 - Aid for Cancer Research from the Ministry of Health and Welfare (56-8), and by a Grant-in- weeks. Similarly, the antitumor effect of Aid from the Japanese Foundation for Multidisci 4-hydroperoxy-CAP was somewhat greater plinaryTreatment of Cancer. The authors would at 1 week than at 3 weeks. However, the like to thank Dr. Yukio Shimosato, Chief of the magnitude of difference in the antitumor Pathology Division, National Cancer Center Re searchInstitute for his valuable comments on this effect of CPA between 1 week and 3 weeks, paper, and Dr. Kazuyoshi Maejima, Chief of the was greater than that in the case of 4-hy Experimental Animal Center of our University droperoxy-CPA. These results may reflect for his cooperation in keeping our test animals. the decrease in the enzyme activity in tumor Thanks are also due to Ms. Shoko Koizumi and bearingnude mice at 3 weeks after tumor Ms. Mariko Ikeda for their technical assistance. inoculation. (ReceivedJan. 6, 1983/AcceptedMarch 24, 1983) However the difference in the effect of

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444 Gann